Menguito C A, Papaconstantinou J, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0647.
Prep Biochem. 1993 Nov;23(4):449-72. doi: 10.1080/10826069308544569.
We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene (hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of approximately 18% of total cell protein (approximately 50 mg/liter of bacterial culture). The fusion HisRS was purified to > 99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the functional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M(r) = 53.7 kDa.
我们描述了马链球菌组氨酸 - tRNA合成酶基因（hisS）在大肠杆菌中的高水平表达以及该基因产物的纯化和特性鉴定。由于hisS基因中缺乏有效的大肠杆菌核糖体结合序列，其编码区与表达载体pT7 - 7进行了读框融合，从而构建了一个融合基因构建体（pT7 - 7recIII），该构建体受强噬菌体T7启动子的控制。另一个构建体（pT - 7recII）用于天然组氨酸 - tRNA合成酶（HisRS）的低水平表达。将这些质粒电穿孔导入已含有pGP1 - 2的大肠杆菌HB101中。温度诱导后，在起始甲硫氨酸和第二个氨基酸赖氨酸之间有额外15个氨基酸的融合HisRS以约占总细胞蛋白18%的水平表达（约50 mg/升细菌培养物）。通过对S100组分进行阴离子交换和阳离子交换色谱相结合的方法，将融合HisRS纯化至> 99%。天然蛋白和融合蛋白的预测分子量分别为47,932和49,717。通过Sephacryl S - 200凝胶过滤色谱法估计活性融合HisRS的质量为94,000 Da，通过非变性聚丙烯酰胺凝胶电泳估计为108,200 Da。这两种方法均表明功能性酶是由两个相同亚基组成的二聚体。对纯化的融合HisRS进行还原或不还原的SDS - 聚丙烯酰胺凝胶电泳分析均显示一条分子量为53.7 kDa的条带。
